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Expression of c-Fos protein in interneurons and


projection neurons of the rat spinal cord in
response to noxious somatic, articular, and
visceral stimulation
ARTICLE in THE JOURNAL OF COMPARATIVE NEUROLOGY AUGUST 1989
Impact Factor: 3.23 DOI: 10.1002/cne.902850203 Source: PubMed

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THE JOURNAL OF COMPARATIVE NEUROLOGY 286177-195 (1989)

Expression of cfos Protein in Interneurons


and Projection Neurons of the Rat Spinal
Cord in Response to Noxious Somatic,
Articular, and Visceral Stimulation
D. M E R E Y , A. GANNON, J.D. LEVINE, AND A.I. BASBAUM
INSERM, U-161, Paris, France (D.M.); and Departments of Anatomy, (A.G., A.I.B.),
and Physiology, (J.D.L.), University of California, San Francisco, California 94143

ABSTRACT
This study used immunocytochemistry to examine the pattern of noxious-stimulus evoked expression of the proto-oncogene c-fos in the spinal cord
of the rat. Both noxious somatic and joint stimulation in awake rats evoked
the expression of c-fos protein in similar areas of the lumbar spinal cord. Cfos-immunoreactive neurons were found in laminae I and outer 11, in the lateral part of the neck of the dorsal horn, and in laminae VII, VIII, and X. All of
the labelled neurons were located ipsilateral to the injured hindpaw, except for
lamina VIII where bilateral labelling was recorded. The c-fos-immunoreactive
neurons in lamina I extended from the L3 segment to the rostral sacral cord;
staining in outer lamina I1 was only found at the L, segment. The more deeply
located cells, of the dorsal and medioventral horns, had the most extensive rostrocaudal spread; they were found from L, through the rostral sacral segments.
The pattern of c-fos-immunoreactivity produced by visceral stimulation,
in anesthetized rats, differed in several ways from that produced by somatic
stimulation. First, there was considerable bilateral, symmetrical labelling of
cells. Second, there was a much more extensive rostrocaudal spread of the
labelling, from cervical through sacral cord. Third, the greatest rostrocaudal
spread was found for neurons in the superficial dorsal horn; labelled cells in
the neck of the dorsal horn and in lamina X were restricted to segments a t the
thoracolumbar junction, which is also where the superficial dorsal horn cells
were most concentrated. Fourth, there were very few labelled neurons in the
outer part of the substantia gelatinosa.
To determine whether any neurons that express the c-fos protein in
response to noxious stimulation project to supraspinal sites, we combined the
immunocytochemical localization of c-fos with the localization of a retrogradely transported protein-gold complex that was injected into the thalamic
and brainstem targets of the major ascending spinal pathways. In rats that
received the somatic noxious stimulus, 90% of all of the c-fos projection neurons were recorded in four major areas of the cord: lamina I (37 % ), the lateral
part of the neck of the dorsal horn (24%),laminae VIII (9%),and X (29%).
The remainder were scattered throughout the spinal gray. With the exception
of lamina VIII, which contained c-fos projection neurons contralateral to the
inflamed paw, all of the c-fos projection neurons were located ipsilateral to the
injured paw. Although c-fos-immunoreactive neurons and retrogradely labelled cells were found in many other areas of the spinal gray that contain

Accepted February 24,1989.


Address reprint requests to Allan I. Basbaum, Department of Anatomy,
University of California San Francisco, San Francisco, CA 94143.

0 1989 ALAN R. LISS, INC.

178

D. MENETREY ET AL.
nociresponsive neurons, few were double-labelled. Finally, retrogradely labelled cells that expressed c-fos in response to visceral stimulation were only
found in the superficial dorsal horn. They were distributed from cervical
through sacral levels; most were at the thoracolumbar junction.
This study demonstrates that the c-fos protein can be used as a functional
marker to identify the spinal neurons that are activated by different forms of
noxious stimulation and indicate that in the awake, freely moving animal,
activity in projection neurons of four regions, lamina I, the lateral neck of the
dorsal horn, laminae VIII and X, contribute to the central transmission of
nociceptive messages that are probably involved in the conscious appreciation
of pain.
Key words: c$os protein, pain mechanisms; immunocytochemistry, retrograde tracing, spinal cord ascending tracts

Electrophysiological studies have characterized two


classes of spinal nociceptive neuron: nociceptive specific
cells (Class 31, which are exclusively driven by noxious
peripheral stimulation, and wide dynamic range cells (Class
21, which are excited by both nonnoxious and noxious
peripheral stimuli. These nociceptive neurons cluster into
three major regions of the spinal cord gray matter. The
superficial dorsal horn (laminae I and outer 11) contains
both Class 2 and 3 neurons; most of the nociceptive neurons
of the neck of the dorsal horn are Class 2; those of the
medioventral horn (including laminae VIII, X and medial
VII) are of the Class 3 variety (Willis, 85; Besson and
Chaouch, 87). Neurons in these areas are at the origin of
five major ascending pathways that are presumed to be
important in nociception (Menktrey, 87). These are the spinosolitary tract, which terminates in the nucleus of the solitary tract; the medial and lateral components of the spinoreticular tracts, which respectively terminate in the medullary
nucleus reticularis gigantocellularis and in the region of the
lateral reticular nucleus; the spinomesencephalic tract,
which terminates in the periaqueductal gray and the parabrachial and cuneiform nuclei of the rostral, dorsolateral
pons; and the spinothalamic tract, which terminates in different subnuclei of the thalamus.
Although there is extensive information about the anatomy, physiology, and pharmacology of the nociceptive messages transmitted by and the inhibitory controls exerted
upon spinal nociceptive neurons, there are important limitations to these studies. First, most of the electrophysiological studies that characterized nociceptive neurons were performed in anesthetized, or decerebrate and/or spinalized
animals. The analysis of the properties of dorsal horn neurons in awake animals has been particularly difficult
(Bromberg and Fetz, 77; Hayes et al., 81;Collins, 87; Duncan et al., 87; Sorkin et al., 88).Second, the noxious stimuli
used were usually of short duration, such as pinprick, pinch,
or noxious heat. Only rarely have recordings been made in
animal models of tonic, or persistent, pain (Menktrey and
Besson, 82; Calvin0 et al., 87; Dickenson and Sullivan, 87).
Third, sample size is very limited in electrophysiological
studies. Although anatomical techniques are more suited to
studying large populations of neurons, the functional properties of labelled neurons cannot be appreciated.
Recently, Hunt et al. (87) demonstrated that it is possible
to monitor the activity of nociceptive neurons of the dorsal horn, with single cell resolution, by using immunocytochemical localization of the protein product of the c-fos

proto-oncogene. C-fos is the cellular homologue of the viral


oncogene, v-jos. Expression of the gene is rapid; c-jos messenger RNA can be detected within 15 minutes of presentation of an appropriate inducing stimulus. The mRNA product, the fos protein, is rapidly synthesized and translocated
to the nucleus (Greenberg and Ziff, 84; Kruiger et al., 84,
85; Greenberg et al., 85, 86; Curran and Morgan, 86; Ran
et al., 86)where it can be localized with antisera. Hunt et al.
(87) reported that the distribution of c-fos-immunoreactive
neurons evoked by different types of peripheral stimulation
was consistent with the known distribution of nociceptive
and nonnociceptive neurons in the dorsal horn.
In this study we extended those observations, by examining the expression of c-fos in response to relatively selective
noxious chemical stimulation of somatic, articular and visceral structures. We also determined whether c-fos is expressed in spinal neurons that project to the brain. This is
important if one is to use the expression of c-jos to monitor the activity of neurons that contribute to the rostrad
transmission of nociceptive messages in the CNS. To address this question we have used a double-labelling method
to localize noxious stimulus-evoked c-fos expression in spinal cord neurons at the origin of major ascending pathways.
This method combined immunocytochemical localization of
the expression of c-jos with the localization of a retrogradely
transported protein-gold complex (Basbaum and Menbtrey,
87). The tracer was injected into supraspinal terminal sites
of the major ascending spinal pathways that have been
implicated in nociceptive transmission.

EXPERIMENTALPROCEDURES
Experiments were performed on 30 adult, male Sprague
Dawley rats (300 gm). Eleven rats were used to establish
appropriate parameters of noxious stimulation. Five control
animals were studied; two were unstimulated, freely moving
rats, three received an injection of saline into either the paw,
ankle, or peritoneal cavity (see below). The remaining 14
rats were used to study c-fos immunoreactive ascending
tract cells. In these double-labelling experiments, we first
injected the retrograde tracer and allowed the animals to
recover several days prior to being exposed to the somatic or
visceral noxious stimuli. This experimental design prevented possible contaminating c-jos expression secondary
to the surgical procedure. The rats were later perfused and
the appropriate sections of spinal cord were processed so

179

Cfos PROTEIN IN RAT SPINAL CORD

Fig. 1. This photomontage at the LSlBsegment of the lumbar cord


illustrates the distribution of immunoreactive c-fos neurons produced
by unilateral injection of complete Freund's adjuvant into the plantar
hindpaw. The figure not only illustrates the distribution of labelled neurons ipsilateral to the injected paw, in lamina I (arrowheads), in the reticular neck of the dorsal horn (Ret DH) and around the central canal

(CC), but also illustrates that there is no labelling contralaterally, with


the exception of laminae VIII (see text). A t this level there is no labelling
in the substantia gelatinosa (SG). A higher power photomicrograph of
the lahelling in lamina I can be seen in Figure 3. Calibration bar equals
500 pm.

that we could identify both the retrogradely labelled and the


immunoreactive cells.

of the lateral reticular nucleus (LRN), the medial reticular


formation (nucleus reticularis gigantocellularis, Rgc); the
mesencephalon (MES), including the periaqueductal gray,
parabrachial area, and nucleus cuneiformis, and the ventrobasal complex of the lateral thalamus. These targets were
approached stereotaxically using coordinates from the atlas
of Paxinos and Watson ('86). Since an injection into the
nucleus of the solitary tract (NTS) typically spreads bilaterally, it was omitted in the studies involving unilateral,
subcutaneous, or periarticular stimulation. However, since
the NTS is clearly involved in the processing of visceral
information, we included the NTS injection, which results
in bilateral labelling in the spinal cord (Menittrey and Basbaum, '87), in the visceral studies. The latter was exposed by
ventroflexing the rat's head and incising the dura over the
cisterna magna.

Injection of the retrograde tracer


The retrograde tracer used was a protein-gold complex
consisting of a wheatgerm agglutinin-apohorseradish peroxidase (WGA-apoHRP, Sigma L0390) to which colloidal gold
(-10 nm) was conjugated (Basbaum and Menittrey, '87). In
order to label as many of the projection neurons as possible,
multiple injections were made in each rat. The injections
were targeted at the terminal regions of the five major
ascending pathways that have been implicated in the transmission of nociceptive messages. Although this approach
did not distinguish between the contribution of the individual pathways, it allowed us to identify noxious stimulusevoked c-fos expression in projection neurons using a minimum number of animals.
Animals were anesthetized with sodium pentobarbital (55
mm/kg; ip) and placed in a stereotaxic head holder. Since we
were interested in distinguishing contralateral from ipsilateral projecting pathways, retrograde tracer injections
were made on one side of the brain. We used 20-40 diameter glass micropipettes to unilaterally pressure inject the
tracer (0.5 to 1.0 ~ 1at) each of the following sites: the region

Peripheral stimulation models


Three noxious chemical stimulation models were used to
activate somatic, joint, or visceral nociceptors. Since our
preliminary studies demonstrated that general anesthesia
both reduces the expression of c-fos and alters the spatial
distribution of c-fos immunoreactive neurons at the spinal
cord level (Presley et al., unpublished observations) most of

Fig. 3. These photomicrographs illustrate the location of c-fosimmunoreactive neurons in the superficial layers of the dorsal horn produced by subcutaneous inflammation of the plantar foot (cf. Fig. I). Five

segments are represented, from L, through L5,e Note that there is a


wider rostrocaudal distribution of labelled cells in lamina I than in the
outer part of lamina 11;the latter are restricted to the L, segment.

Fig. 2. These schematics illustrate the distribution of c-fos-immunoreactive neurons in the lumbar cord of a rat that received a unilateral
injection of complete Freunds adjuvant in the right plantar hindpaw, 16
hours prior to being sacrificed. Seven levels are represented, from the L1
through the L6/S1 segments. Each diagram includes all labelled cells in

three 50 Mm sections; each dot represents one labelled cell. Note that
there is a more restricted rostrocaudal distribution of c-fos positive cells
in the superficial dorsal horn than in deeper layers of the spinal gray.
The boundary of the reticular part of the neck of the dorsal horn (Ret.
V) is outlined for orientation.

182

D. MENETREY ET AL.

n
Subcutaneous Inflammation (Plantar Foot)
Tracer Contralateral t o Stimulated Side

L5/6

J3
N

s\

1 rnrn

L6/S 1
Fig. 4. These schematics illustrate the distribution of c-fos-immunoreactive ascending tract cells in the lumbar cord of a rat with unilateral
subcutaneous inflammation of the plantar hindpaw (cf. Fig. 1).The
tracer was unilaterally injected into the thalamus, mesencephalon, lat-

era1 reticular nucleus and reticular formation, contralateral (A) or ipsilateral (B) to the inflamed paw. Each diagram includes double-labelled
cells from five 50 pm sections. Each dot represents one double-labelled
neuron.

183

Cgos PROTEIN IN RAT SPINAL CORD

Subcutaneous Inflammation (Plan tar F o o t )


T r a c e r l p s i l a t e r a l to Stimulated Side

1 mm

Figure 4

184

D. MENETREYET AL.
from:

Lamina I
Ret. DH
Lamina x
S t irnulated
side

S t irnulated
side

from:

Lamina V l l l

A
1

Fig. 5. Schematic representation of the spinal gray matter origin of


the c-10s positive ascending projection neurons. The overall regional
source of doubIe-labelled cells is indicated by stippling; the cell bodies
represent the output from this region, not the output from a particular
lamina. The inflamed paw (stimulated side) is on the left. The upper
diagram illustrates projecting cells that originate in lamina I, the reticu-

lar neck of the dorsal horn (Ret. DH) and lamina X; the lower diagram
illustrates projecting cells that originate in lamina VIII. The thickness of
the arrows is proportional to the numbers of contralaterally and ipsilaterally projecting cells that originate from the particular source. Note
that all double-labelled cells in lamina VIII are located contralateral to
the inflamed paw.

the studies were performed in awake, freely moving animals.


The somatic and joint stimulation protocols were based on
established chronic inflammation models in awake animals.
These protocols were evaluated and approved by the Committee on Animal Research a t UCSF. Somatic stimulation
involved unilateral subcutaneous injection of 150 pl complete Freund's adjuvant into the plantar foot (Ruda e t al.,
'88). The injection was made under brief halothane anesthesia. Periarticular noxious stimulation was produced by
unilaterally placing 150 pg of urate crystals through a skin
incision close to the ankle joint (Otsuki e t al., '86; Coderre
and Wall, '87)- The crystals were implanted under pentobarbital anesthesia (40 mg/kg; ip). The animals were awake
within 2 hours. Periarticular urate crystal injection pro-

duces some inflammation of somatic tissue, but there is also


significant inflammation of the joint. For both somatic and
joint stimulation protocois, the rats were perfused 16 hours
after injection.
The visceral stimulation protocol is based on the aceticacid stretching test (Taber et al., '69) and involves injection
of 0.5 ml of 9% acetic acid into the peritoneal cavity, just o f f
the midline. These studies were performed under general
anesthesia (55 mg/kg; i.p.).* Rats were perfused 1 hour after
injection of the acetic acid. In light of the above comments

'Committee on Animal Research approval t o perform the visceral stimulation studies in awake animals is pending.

Cfos PROTEIN IN RAT SPINAL CORD

185

Tissue processing

Fig. 6. This brightfield photomicrograph illustrates c-fos-immunoreactive and retrogradely labelled neurons in a 40-pm thick horizontal
section through lamina I of the lumbar dorsal horn. C-fos positive cells
have a uniformly stained nucleus (arrowhead). Projection neurons contain punctate cytoplasmic label, which denotes the silver precipitate; the
nuclei of single labelled projection neurons are unstained (open arrow).
Double-labelled cells (arrow) have a densely stained nucleus and cytoplasmic silver precipitate.

concerning the effects of general anesthetic on c-fos immunoreactivity in the spinal cord, it is likely that the numbers
and distribution of cells observed in the visceral stimulation
studies underestimate what would be generated in the
awake animal.
Several control studies were performed. Two unstimulated, freely moving animals were perfused and the spinal
cord examined. Consistent with the report of Hunt et al.
('87), with the exception of a few lightly labelled neurons in
laminae I11 and IV, we found almost no c-fos immunoreactive neurons in the spinal cord. In three additional animals,
we studied the effect of injecting the appropriate volume of
saline, into either the plantar foot, the ankle joint (after skin
incision), or the peritoneal cavity. Two of the rats (plantar
foot and joint) were perfused 16 hours later; the third was
perfused 1 hour after the intraperitoneal injection. The
same number of sections was studied in control and experimental animals; in none of these animals was there significant c-fos expression in the spinal cord. Importantly,
although the skin incision (for urate crystal injection)
evokes the expression of c-fos in the spinal cord, the control
study established that by 16 hours almost no cell labelling
persisted.

Animals were deeply anesthetized and perfused through


the aorta with 200 ml of phosphate-buffered saline (PBS)
followed by 500 ml of 4% paraformaldehyde. The brain and
spinal cord were then removed, and postfixed for 4 hours in
the same fixative a t 4C before they were cryoprotected in
phosphate-buffered 30 % sucrose solution overnight. Serial
frozen sections of the spinal cord (40 hm) or brain (100 pm)
were cut and collected in phosphate buffer. Since the dense
gold deposit at the injection site can be visualized without
silver intensification, brain sections were mounted, stained,
and coverslipped for localizing injection sites. The spinal
cord sections were processed as free-floating sections. They
first underwent a silver intensification procedure to make
the gold visible at the light microscopic level (Danscher, '81)
and then were immunostained with the avidin-biotin procedure of Hsu et al. ('81) by using commercial kits (Vector
Labs, Burlingame, CA). Details of this double-labelling
procedure have been described previously (Menktrey, '85;
Basbaum and MenBtrey, '87). After the DAB reaction was
completed, the sections were air dried, mounted, and coverslipped. The location of retrogradely labeled cells (containing silver particles) and c-fos-immunoreactive neurons were
plotted with a camera lucida attachment. The location of
cells described is based on the spinal cord cytoarchitectonic
atlas of Molander et al. ('84).
Four different primary antisera were tested. Most of the
studies were performed with a rabbit polyclonal antiserum,
directed against an in vitro translated c-fos gene product,
kindly provided by Dr. Dennis Slamon (Department of
Medicine a t UCLA). We also used three monoclonal antisera (Microbiological Associates Inc., Bethesda) that were
raised against synthetic peptide fragments from different
regions of the c-fos protein, residues, 4-17 (N terminal),
132-154 (mid) and 359-378 (C terminal). Good results were
only obtained with the polyclonal antiserum and the N terminal directed monoclonal antiserum. Both of these antisera were used at a 1/5,000 dilution, with the polyclonal
antiserum preabsorbed against acetone-dried liver powder
prior to use. The two antisera revealed the same pattern of
staining; however, the immunoreactivity was always more
intense with the polyclonal antiserum.
The c-fos protein is not available in sufficient quantities
and thus absorption controls could not be performed with
the polyclonal antiserum, which was directed against the
entire c-fos gene product. Preabsorption of the N-terminal
directed monoclonal antiserum with synthetic N-terminal
peptide, (1 wg/ml diluted antiserum), however, completely
abolished the staining. The staining pattern was not
changed when the N-terminal monoclonal antiserum was
absorbed with either the synthetic C-terminal peptide or
peptide from the midportion of the c-fos protein.

RESULTS
Characteristics of C@x-immunoreactive
neurons
As described above, with the exception of a few very
lightly labelled cells in laminae I11 and IV, there were no cfos-immunoreactive spinal neurons in control rats. C-fosimmunoreactive cells were found only in the spinal cord of
rats that experienced a peripheral noxious stimulus (Fig. 1).
In this respect, the spinal cord can be distinguished from
certain brainstem and forebrain areas where we have

186

D. MENETREY ET AL.

Fig. 7. These photomicrographs illustrate c-fos-immunoreactive


and retrogradely labelled neurons in a 40-gm thick horizontal section
through the reticular neck of the dorsal horn. With darkfield illumination (1)the longitudinal bundles of fibers in the reticular part of the dorsal horn (Ret V) can be distinguished from the more medial dorsal horn
(mDH). There are large numbers of retrogradely labelled cells (bright

dots) in the reticular neck; some are immediately adjacent to the dorsolateral funiculus (DLF). The dorsal columns (DC) are on the left. The
higher magnification brightfield photomicrographs (2 and 3) illustrate
the two double-labelled neurons that are indicated by arrows in 1. Calibration bars equal 50 pm.

recorded a baseline level of neuronal c-fos-immunoreactivity. In the case of rats with hindpaw injury, the c-fos positive
neurons were found almost exclusively ipsilateral to the
injured paw and at rostrocaudal levels of the spinal cord
that receive afferent fibers from the injured limb (Devor and
Clayman, '80; Molander and Grant, '85; Swett and Woolf,
'85). Importantly, since the retrogradely labelled neurons
were found at all levels of the spinal cord, but the c-fos
expression was restricted to the lumbosacral cord, it is clear
that the c-fos-immunoreactivity was not secondary to the
neuron having transported the retrograde tracer. Moreover,
with the same noxious stimulus, the pattern of c-fos immunoreactivity was the same, whether the animal received the
retrograde tracer or not; thus the pattern of c-fos expression
is not a function of some interaction between the retrograde
tracer and the noxious stimulus.
The c-fos-immunoreactive material was uniformly distributed in the nucleus of lahelled neurons; nucleoli were
not labelled. This staining pattern and the overall distribution of labelled cells was the same whether we used the polyclonal or the N-terminal directed monoclonal antisera. Although the monoclonal antibody only stained neuronal cell
nuclei, there was some additional staining seen with the
polyclonal antiserum. Specifically, we sometimes noted a
filamentous, cytoplasmic staining in some small cells that
surround the central canal; less commonly, we noted cytoplasmic staining in a few cells of the substantia gelatinosa

and in the lateral spinal nucleus of the dorsolateral funiculus. Immunostained terminals were occasionally recorded in
the superficial dorsal horn. This presumed crossreactivity
was readily distinguished from the diffuse, nuclear c-fosimmunoreactivity.

Distribution of ascending tract cells in the


spinal cord
Consistent with the known cells of origin of the brainstem
and thalamic targets injected with retrograde tracer, the retrogradely labelled spinal projection neurons clustered into
several distinct populations (see references in Leah et al.,
'88). At all lumbar segments, projection neurons were
located in lamina I of the dorsal horn, in the reticular part of
the neck of the dorsal horn, in laminae VII, VIII, and X, and
in the lateral spinal nucleus of the dorsolateral funiculus.
Two additional clusters of cells were labelled only at upper
lumbar segments. One cluster was a t the medial border of
the reticular part of the neck of the dorsal horn; this is
another source of spinal projections to the region of the lateral reticular nucleus (Menktrey et al., '83). The second cluster was located in the most ventromedial part of the dorsal
horn, abutting the dorsal columns; this region contains cells
at the origin of spinothalamic tract axons (Giesler et al., '79;
Menktrey et al., '84b). The majority of the retrogradely
labelled cells were located contralateral to the injection site;
however, a substantial number (30%) were located ipsi-

187

C f o s PROTEIN IN RAT SPINAL CORD

lateral. The latter are primarily cells at the origin of the


spinoreticular projections, which are bilaterally projecting
systems (Menbtrey, '87). This widespread distribution
indicates that there was significant transport from all sites
injected with the retrograde tracer; however, since not all
terminal sites could have been injected, the numbers of retrogradely labelled cells, and thus the number of doublelabelled neurons (see below) is certainly underestimated.

Subcutaneous inflammation

Fig. 8. These photomicrographs were taken from a 40-pm horizontal


section that was located slightly dorsal to the section in Figure 6. They
illustrate retrograde labelling in the medial neck of the dorsal horn
(mDH). This cluster of retrogradely cells has a very restricted dorsoventral and rostrocaudal distribution; neurons in this region primarily project to the region of the lateral reticular nucleus (MenBtrey et al., '83).
Although c-fos-immunoreactive neurons are found in this region ( l ) ,
double-labelled cells were rarely found. The higher magnification
hrightfield photomicrograph (2) illustrates the double-labelled neuron
that is indicated hy an arrow in 1. The cell is located laterally, in the
reticular neck (Ret V) of the dorsal horn. DLF and DC identify the dorsolateral funiculus and dorsal columns, respectively. Calibration bar
equals 50 pm.

C-fos expression was examined 16 hours after unilateral


injection of 150 pl complete Freund's adjuvant in the plantar surface of one hindpaw. The subcutaneous inflammation
that resulted extended over the plantar surface of the paw,
the toes, and the tissue surroundingthe ankle. Figure 2 illustrates the distribution of c-fos-immunoreactive neurons in
the spinal cord of a rat injected with adjuvant. Labelled neurons were found from the L, to L, spinal segments and in the
rostral sacral cord. All but a few were ipsilateral to the adjuvant injection. The density of staining varied over the rostrocaudal extent of the cord, with the most densely labelled
nuclei located close to the entry zone (L4and L5)of the afferent fibers that innervate the stimulated area. Lightly
labelled neurons were most commonly found rostral and
caudal to the L4,5segments. They were also found intermingled with the heavily labelled cells of the L, and L, segments. The greatest concentration of ipsilateral c-fosimmunoreactive neurons was in the superficial layers of the
dorsal horn, laminae 1and outer 11, in the reticular part of
the neck of the dorsal horn, around the base of the dorsal
horn, and in laminae VII, VIII, and X. Some cells were
found in the lateral spinal nucleus. In some rats, a few cells
were found in the nucleus proprius, laminae I11 and IV, in
the inner part of the substantia gelatinosa, in the medial
part of the neck of the dorsal horn (i.e., medial lamina V), or
in lamina IX.
Significant differences exist in the rostral caudal extent of
the labelling in these different laminae (Fig. 2). The labelled
neurons in outer lamina I1 were the most restricted rostrocaudally; they were only found at the L4 segment (Fig. 3).
The staining in lamina I, however, extended from the L, segment to the rostral sacral cord. Labelled cells in lamina I of
the rostral lumbar segments were concentrated medially;
they shifted laterally in more caudal sections. Cells of the
reticular part of the dorsal horn and in laminae VII, VIII,
and X had the most extensive rostrocaudal spread, from all
levels of the lumbar cord to the most rostral sacral segments.
Finally, some c-fos-immunoreactive neurons were found in
lamina VIII contralateral to the side of the adjuvant injection.
C-fos-immunoreactive projection neurons. The
distribution of contralaterally and ipsilaterally ascending cfos-immunoreactive projection neurons was studied in rats
that received unilateral tracer injections, either contralatera1 or ipsilateral to the inflamed paw. Although it is of
interest to determine the relative proportion of contralaterally and ipsilaterally projecting c-fos-immunoreactive cells
in the same animal, we avoided this approach since it would
have required that the rats be injected bilaterally with adjuvant. The tracer injections were made in the thalamus, midbrain, nucleus reticularis gigantocellularis, and the region of
the lateral reticular nucleus. As described above, since injections into the nucleus of the solitary tract typically spread
bilaterally, that injection was omitted for these studies. The
counts of single and double-labeled cells were made in the

188

D. MENETREY ET AL.

Fig. 9. This brightfield photomicrograph is from a 40-pm transverse


section through lamina X of the lumbar spinal cord. Arrowheads point
to c-fos immunoreactive cells (with uniform, densely stained nucleus).
Retrogradely labelled cells have punctate cytoplasmic silver precipitate

and a clear, unstained nucleus. The open arrows point to retrogradely


labelled, but not immunoreactive neurons. The arrows point to doublelabelled cells that are located just dorsal to the central canal (CC) and
ventral to the border (dotted line) of the dorsal columns (DC).

L,-L, segments, which contained the majority of the doublelabelled cells.


Figure 4 illustrates cases in which the retrograde tracer
was injected contralateral (A) or ipsilateral (B) to the
inflamed hindpaw. Double-labelled cells were commonly
observed in all rats that received tracer injections. Figure 5
schematizes the distribution of these contralaterally and
ipsilaterally projecting c-fos-immunoreactive neurons. All
but some in lamina VIII were located ipsilateral to the
injected hindpaw. The rostrocaudal location of the doublelabelled cells included all lumbar spinal segments; the maximum concentration (82%) was found at segments L,-L5.
The double-labelled cells constituted only a small proportion of the total population of c-fos-immunoreactive neurons and the total populat,ion of ascending tract cells. The
mean values were 6% and 8 % , respectively. Despite the
wide distribution of both c-fos positive and ascending tract
cells, however, 90% of the double-labelled cells were clustered in four discrete areas: laminae I and outer 11, the lateral, reticular part of the neck of the dorsal horn, lamina
VIII, and lamina X.
The superBcia1 dorsal horn. Double-labelled cells
were most commonly recorded in the superficial dorsal
horn. They constituted 37% of all double-labelled cells in
the spinal cord. Almost all were located in segments L,-Ls.
Very few were found at L, and L,, where the c-fos positive
cells were located medial to the bulk of the retrogradely
labelled neurons. All but a few of the superficial dorsal horn
double-labelled neurons were in lamina I; the remainder
were located in outer I1 and were confined to the L4,6junction. Approximately 16% of all the retrogradely labelled
cells and 19% of all the c-fos positive cells in the superficial
dorsal horn at segments L,-L, were double-labelled. Figure
6 illustrates examples of double-labelled cells in lamina I.
C-fos-immunoreactive neurons in lamina I contribute to
both contralateral and ipsilateral ascending pathways. Rela-

tive to the total population of contralaterally or ipsilaterally


projecting cells, we found a greater number of superficial
dorsal horn neurons that projected contralaterally. Thus
46 % of all c-fos positive contralaterally projecting tract
cells, but only 25% of all c-fos positive ipsilaterally projecting tract cells were located in the superficial dorsal
horn.
The reticular part of the dorsal horn. C-fos-immunoreactive projection neurons were also common in the lateral, reticular, part of the neck of the dorsal horn (Figs. 7 , 8 ) .
All double-labelled cells in this part of the cord were located
ipsilateral to the inflamed paw; most were located in the LaL, segments. These cells constituted 24% of all doublelabelled cells recorded in the spinal cord. In contrast to the
cells located in the superficial dorsal horn, they were less
common in contralateral than in ipsilateral projecting pathways (17% of all c-fos positive contralateral tract cells vs.
32% of all c-fos positive ipsilateral tract cells). Approximately 10% of all the retrogradely labelled cells and 11% of
all the c-fos positive cells in the lateral part of the neck of
the dorsal horn were double-labelled. The clustering of double-labelled cells in this region distinguishes it from the
medially and ventrally adjacent grey matter that contains
both c-fos and retrogradely labelled neurons, but very few
double-labelled cells.

Fig. 10. These schematics illustrate the distribution of c-fos-immunoreactive cells in the lumbar cord of a rat that experienced periarticular inflammation after implant of urate crystals close to the ankle. The
rat was perfused 16 hours after the crystals were implanted. Seven levels
of the cord are represented, from the L1through the Ls/S, segments.
Each diagram includes all labelled cells in three 50-um sections: each dot
represeni$ one labelled cell.

Ankle joint urate arthritis

Figure 10

D. MENETREY ET AL.

190

Visceral Stimulation
(anesthetized)

L6/S1
Figure 11

Visceral Stimulation
(anesthetized)

Thor

L4

1 mm

L6/S1

Fig. 12. These schematics illustrate the distribution of c-fos-immunoreactive ascending tract cells in
response to visceral stimulation. The retrograde tracer was injected into the thalamus, mescencephalon, lateral reticular nucleus, and reticular formation unilaterally and into the nucleus of the solitary tract; the latter injection spread bilaterally. Each diagram includes double-labelled cells from five 50 pm sections (cells
from both sides of the cord are plotted). Each dot represents one double-labelled neuron.
Fig. 11. These schematics illustrate the cervical (C,) through lumbosacral (LJS,) distribution of c-fos-immunoreactive cells in response to
noxious visceral stimulation (intraperitoneal injection of 0.5 ml of 9%
acetic acid). The experiment was performed under general anesthesia
and the rat was perfused 1hour after visceral stimulation. Each diagram
includes all labelled cells in three 50-pm sections; each dot represents
one labelled cell. Since we found bilaterally symmetric labelling after

visceral stimulation, we have only illustrated the cell distribution on one


side of the cord. The boundary of the reticular part of the neck of the
dorsal horn is outlined for orientation. Note that the densest staining is
at the thoracolumbar junction (T13-LJ and the most extensive rostrocaudal distribution of labelled cells was found in the superficial dorsal
horn.

192

D. MENETREY ET AL.

The are- surrounding the central canal. C-fos- found after adjuvant injection only in that fewer doubleimmunoreactive projection neurons were numerous in the labelled cells were recorded.
area surrounding the central canal. They were found in lamVisceral noxious stimulation
ina X throughout the lumbar enlargement and at the LZw3
levels, in a region located just dorsolateral to lamina X. The
The entire visceral stimulation protocol, which lasted 1
double-labelled cells in the region of the central canal con- hour, was carried out under general anesthesia. The pattern
stituted approximately 20% of all c-fos positive projection of c-fos staining (Fig. 11) was significantly different from
neurons recorded in the lumbar cord. All of these double- that found after subcutaneous or periarticular inflammalabelled cells were located ipsilateral to the inflamed paw tion in the awake rat. First, visceral stimulation produced a
and were approximately equally divided between contralat- much more bilaterally symmetric distribution of c-foserally and ipsilaterally projecting pathways. Figure 9 illus- immunoreactive neurons, perhaps because of the bilateral
trates examples of double-labelled cells in lamina X.
nature of the stimulus (intraperitoneal injection). Second,
Lamina WII. Double-labelled cells in lamina VIII ac- the c-fos labelling was not restricted to the lumbar enlargecounted for 9% of all c-fos positive projection cells recorded ment, but extended from cervical to sacral cord. The
in the lumbar spinal cord. As in the region of the central greatest density and largest number of labelled cells was,
canal, contralaterally and ipsilaterally projecting c-fos posi- however, found a t the thoraco-lumbar junction, segments
tive cells were equally divided. In contrast to the other dou- T,,-L,. Third, the number of labelled cells in the superficial
ble-labelled cells, however, all c-fos positive projection neu- dorsal horn was much greater than in deeper areas (77% vs.
rons in lamina VIII were located contralateral to the 23 % of all c-fos-immunoreactive cells, respectively).
inflamed paw. None of the c-fos-immunoreactive neurons in Fourth, there was a significant difference in the rostrocaulamina VIII ipsilateral to the inflamed paw were double- dal extent of the labelled cells located superficially from
labelled. Thus the ipsilaterally projecting c-fos positive lam- those located in deeper areas. The labelled neurons in the
ina VIII cells terminated in the brain contralateral to the superficial dorsal horn were the only ones located at cervical
injured paw; those projecting contralaterally terminated ip- through sacral levels; the c-fos-immunoreactive neurons in
silateral to the injured paw. The presence of the latter path- the neck of the dorsal horn and in lamina X were confined to
way suggests that information from the inflamed paw may, the thoracolumbar junction. At most levels the marginal
in part, access the brain ipsilateral to the injury, via a neural cells spanned the whole mediolateral extent of the gray matnetwork that crosses the cord at least twice.
ter; at cervical levels, the labelled neurons were located
Other spinal areas. Double-labelled cells in the re- along the lateral border of the gray matter.
maining areas of the spinal cord accounted for a t most 10%
Figure 12 illustrates the distribution of c-fos positive
of all the spinal c-fos positive projection neurons. These ascending tract cells in the spinal cord of a rat that received
double-labelled cells were scattered along the lumbar en- a unilateral tracer injection in the thalamus, midbrain,
largement in several areas, including the medial part of the nucleus reticularis gigantocellularis, and lateral reticular
neck of the dorsal horn, the intermediate gray between the nucleus contralateral to the side of the acetic acid injection
dorsal and ventral horns, and the lateral spinal nucleus of and into the nucleus of the solitary tract. As described
the dorsolateral funiculus.
above, the NTS injection typically spreads bilaterally. Although the c-fos-positive projection cells were located bilatPeriarticdar inflammation
erally, we plotted all double-labelled cells on one side. DouC-fos expression was examined 16 hours after unilaterally ble-labelled neurons were found at upper thoracic through
implanting 150 kg of urate crystals close to the ankle joint. sacral spinal levels, but were most concentrated at the thoThe inflammation that resulted extended over the tissue raco-lumbar junction. No double-labelled cells were resurrounding the ankle as well as the proximal part of the corded at cervical levels. All but a few of the c-fos-immunodorsum of the foot. The severity and extent of the inflam- reactive projection neurons were located in lamina I of the
mation was much less than was seen with adjuvant injection. superficial dorsal horn; occasional cells were recorded in the
Figure 10 illustrates the resulting pattern of c-fos-immuno- white matter adjacent to the neck of the dorsal horn.
reactivity in these animals. C-fos labelled cells were found Although c-fos-immunoreactive and retrogradely labelled
from segments L1 through L,; almost all were located ipsi- neurons were found in lamina X, none of those were doublelateral to the inflamed paw, in lamina I, the lateral neck of labelled.
the dorsal horn, at the base of the dorsal horn, and in laminae VII, VIII, and X. Very few labelled cells were recorded
DISCUSSION
in the outer part of the substantia gelatinosa or in contralatConsistent with the original report of Hunt et al. ('87), we
era1 lamina VIII. Thus with the exception of minor differences in the superficial dorsal horn and the minimal contra- found that some spinal neurons express c-fos in response to
lateral neuronal labelling, the pattern of staining after urate noxious peripheral stimulation. We also demonstrated that
crystal implantation was comparable to that seen after different patterns of c-fos-immunoreactivity are produced
injection of Freund's adjuvant. The major difference was by stimuli targeted a t different peripheral structures and
quantitative; that is, many more neurons were found after that some of those neurons that express c-fos are a t the oriadjuvant injection, which suggests that the numbers of cells gin of major ascending spinal pathways, many of which have
labelled is related to the severity of the inflammation.
been implicated in the rostra1 transmission of nociceptive
We used the protocol described above to study the distri- messages.
bution of contralaterally and ipsilaterally projecting c-fos
T o study noxious stimulus-evoked expression of c-fos, we
positive ascending tract cells. Double-labelled neurons were chose two subacute inflammation models and one acute visseen in both series of experiments and were concentrated in ceral stimulation model. These stimulation protocols generlamina I, the reticular neck of the dorsal horn, and to a lesser ated considerable labelling in laminae I and outer 11, the
extent in lamina X. This distribution differed from that neck of the dorsal horn, and in laminae VII, VIII and X of

C f o s PROTEIN IN RAT SPINAL CORD

the ventromedial gray. Electrophysiological studies have established that all of these spinal areas contain nociceptive
cells, many of which are strongly driven under conditions of
inflammation (Menbtrey and Besson, '82; Calvino et al., '87;
Dickenson and Sullivan, '87). It was particularly relevant
that we found dense labelling of neurons in laminae I and
outer 11, but rarely in the inner part of the substantia gelatinosa, a region that predominantly is activated by nonnoxious stimulation (Light et al., '79; Bennett et al., '82). Thus
the distribution of c-fos-immunoreactive neurons was essentially restricted to the known distribution of nociceptive
spinal neurons. However, since neither the factors that
induce c-fos expression nor the consequences of expression
of the c-fos protein are known, caution must be taken in
interpreting these data. We cannot conclude that all
labelled cells are nociceptive nor can we conclude that a particular neuron was transmitting a nociceptive message. The
absence of induction of c-fos in a spinal neuron also does not
mean that the neuron was not nociceptive. The neuron may
not synthesize c-fos, or may only do so in amounts too low to
be detected by immunocytochemistry. In other words, the
c-fos protein is merely a marker that tells us that something
happened to that particular neuron in response to the
applied peripheral stimulus. In spite of these caveats, we
believe that this technique allows one to monitor, with single cell resolution, the responsiveness of large populations of
neurons to noxious stimulation in awake animals, i.e., in situations in which the pain behavior of the animal can simultaneously be evaluated. Although the 2-deoxyglucose
method (Sokoloff et al., '77) has been used to monitor functional changes in the spinal cord (Ciriello et al., '82; Abram
and Kostreva, '86), the resolution of the technique is poor
and the approach cannot be combined in double-labelling
(i.e., projection neuron) studies. The cytochrome oxidase
method offers cellular resolution but has yet to be used to
map noxious-stimulus evoked changes in metabolic activity
of spinal cord neurons (Wong-Riley and Kageyama, '86).
Baseline levels of cytochrome oxidase activity are high,
which may make it difficult to detect noxious stimulusevoked increases in enzyme activity.
This study revealed several important features of the spatial organization of spinal nociceptive transmission systems.
First, there is a very widespread rostrocaudal distribution of
neurons that respond to a relatively localized peripheral
noxious stimulus. Second, there is a differential laminar distribution of responsive neurons a t different rostrocaudal
levels, and third, there is a differential pattern of responsive
neurons produced by subacute somatic or periarticular inflammation and acute, visceral stimulation.
Subcutaneous or periarticular inflammation produced
neuronal staining through all lumbar segments into the rostral sacral cord; it thus included several segments rostral to
the region (L3-L5),which receives the densest primary afferent fiber input from the stimulated paw. The laminar distribution of labelling was extensive and included the superficial dorsal horn, which contained the densest concentration
of cells, the lateral part of the neck of the dorsal horn, the
intermediate gray (i.e., laminae VI and VII), and laminae
VIII and X. It is of interest that there were nonrandom variations in the intensity of staining throughout the lumbar
cord. In general the most densely stained cells were located
in the L,-L, segments. The paler staining of neurons located
rostrally suggests that the density of staining is proportional
to the amount of afferent drive.

193

The most restricted rostrocaudal distribution of labelled


cells was found in the superficial laminae. It appears that
the segmental pattern of labelling in laminae I and outer I1
follows the central somatotopic organization of the cutaneous afferent fibers that innervate the inflamed area (Devor and Claman, '80; Molander and Grant, '85; Swett and
Woolf, '85). Specifically, the progressive medial shift in the
location of c-fos-immunoreactive neurons in lamina I and
their dropout at more rostral lumbar segments parallelled
the central projection of small diameter primary afferents
from the hindpaw. These data suggest that the cells of the
superficial dorsal horn that express c-fos receive a monosynaptic input from the small diameter afferent fibers that
innervate the inflamed area. The most widespread rostrocaudal distribution of labelled cells, however, was found in
laminae VII and VIII. Since small diameter, nociceptive primary afferents do not terminate in these regions (Light and
Perl, '79; Suguira et al., '87), it is likely that the nociceptive
input to cells in laminae VII and VIII is polysynaptic. Furthermore, since single cells in laminae VII and VIII have
rather large cutaneous receptive fields (Fields et al., '75, '77;
Giesler et al., '81; Cervero and Wolstencroft, '84; Men6trey
et al., '84a), the polysynaptic input probably derives from
relatively large regions of the body. Taken together, these
data could account for the widespread rostrocaudal distribution of c-fos immunoreactive neurons in deeper parts of
the spinal cord.
The pattern of c-fos expression produced by visceral stimulation differs significantly from that seen after subacute
inflammation of the paw. The injection into the peritoneal
cavity produced extensive rostrocaudal labelling, from the
cervical through sacral cord. Since there was probably
spread of the injection volume, it is possible that some of the
labelling in the cervical cord reflected activation of diaphragmatic afferents. The extensive pattern of labelling
may, however, also relate to the fact that small diameter primary afferents that innervate visceral structures arborize
over more segments than do cutaneous or muscle afferents
(Neuhuber, '82; Neuhuber et al., '86; Suguira, submitted).
Interestingly, this extensive distribution was found only for
cells in the superficial dorsal horn; deeper cells (in the neck
of the dorsal horn and in lamina X) were confined to the
thoracolumbar junction. Importantly, although the anesthetic conditions under which the visceral experiments were
run may have restricted the c-fos labelling pattern (Presley
et al., unpublished observations), our results are consistent
with anatomical and electrophysiological studies that have
implicated neurons in laminae I and X in the central transmission of visceral nociceptive information in the rat (Takahashi and Yokota, '83; Ness and Gebhart, '87). In fact, the
very limited expression of c-fos of neurons in the substantia
gelatinosa after acetic acid injection is consistent with the
sparse projection of visceral afferents to lamina I1 (Cervero
and Connell, '84; Morgan et al., '81).
The great advantage of a functionally oriented anatomical approach is that the "activity" of large numbers of neurons can be readily identified. Although electrophysiological
studies can unequivocally characterize the nociceptive
properties of spinal cord neurons, it is both difficult and
time-consuming to locate a large sample of physiologically
characterized projection neurons that can be antidromically
activated. Monitoring the noxious stimulus-evoked expression of the c-fos protein in spinal cord projection neurons
thus powerfully complements electrophysiological studies.

194

We found that the c-fos positive ascending tract cells constituted a small percentage of all spinal neurons that
expressed c-fos and of all neurons that were retrogradely
labelled. These data suggest that most of the cells which
express c-fos in response to noxious stimulation make short
intraspinal connections. These may be interneurons or propriospinal neurons. One explanation for the relatively low
percentage of double-labelled neurons is that none of the
ascending pathways studied are purely nociceptive. In fact,
in the basal part of the dorsal horn and medioventral horn of
the rat spinal cord there are many nonnociceptive spinothalamic and spinoreticular neurons that transmit cutaneous or proprioceptive messages (Menktrey et al., 84a,b;
Ness and Gebhart, 87). These are intermingled with the
nociceptive projection neurons and might be retrogradely
labelled, but would probably not express c-fos under the
experimental conditions we used. It is also certain that only
a part of all the nociceptive afferents were activated with the
stimulation parameters used. Thus, for example, any spinal
neurons selectively responsive to noxious stimulation of
muscles or joints would have been only minimally activated
by subcutaneous injection of adjuvant into the hindpaw.
It was highly significant that the double-labelled c-fos
cells were concentrated in very discrete spinal areas. In fact,
the regional distribution of nociceptive cells, defined in
electrophysiological studies, and of ascending tract cells,
defined in anatomical studies (refs. in Menktrey, 87), is
considerably larger than the regional distribution of c-fos
positive ascending tract cells. This indicates that among the
many regions of the cord that contain nociceptive neurons,
the following regions, the superficial dorsal horn, the lateral
neck of the dorsal horn, and laminae VIII and X, may be
particularly relevant to the transmission of nociceptive messages to supraspinal levels via long ascending tracts.
A major goal of pain research studies is to understand the
relative contribution of the different types of nociceptive
neurons, in the different regions of the cord, to the conscious
appreciation of pain. With rare exception, electrophysiological studies are performed in anesthetized or decerebratespinal preparations. Thus the spinal cord neuronal activity
in an animal that experiences pain cannot be evaluated. By
monitoring noxious stimulus-evoked c-fos expression in
projection neurons in awake animals, we can draw some conclusions about the overall pattern of activity generated by
pain-producing stimuli. The superficial dorsal horn contains wide dynamic and nociceptive specific neurons (class 2
and 3, respectively). Most of these have relatively restricted
receptive fields, which are somatopically organized within
the cord. Nociceptive neurons of the lateral part of the neck
of the dorsal horn are predominantly wide dynamic range
cells and have a higher degree of spatial and/or modality
convergence. Neurons in both regions are believed to contribute to the detection and localization of noxious stimuli.
On the other hand, the class 3, nociceptive-specific neurons
of deeper parts of the cord (laminae VII and VIII) have complex excitatory and inhibitory receptive fields that are much
larger and are often bilateral. These neurons are thus probably not involved in the location and discrimination of the
noxious stimulus (as are the dorsal horn nociceptive neurons), but rather have been implicated in the escape reactions and motor responses produced by noxious stimuli.
Since we have shown that ascending projection neurons in
all of these regions are activated under conditions of tonic
pain, the present data indicate that a wide variety of nociceptive projection neurons come into play when an animal

D. MENETREY ET AL.
experiences a tonic noxious stimulus. Since different neurons within the four major regions in which double-labelled
cells were located project to brainstem and thalamus, we
cannot be certain of the specific targets of the projection
neurons that express c-fos. This, however, is the first case in
which it has been demonstrated that these pathways are
indeed activated by noxious stimuli in the awake animal; we
are presently studying the projection of c-fos-immunoreactive neurons to particular brainstem and thalamic loci.
In conclusion, this study provides evidence that c-fos is
expressed in subpopulations of spinal cord cells and that it
can be used as a functional marker for populations of nociceptive cells, including those at the origin of long ascending
tracts. The findings of this study emphasize the contribution of neurons in the superficial laminae, in the lateral neck
of the dorsal horn, laminae VIII and X to the central transmission of nociceptive information in animals that experience pain. The ability to monitor the activity of large
numbers of nociceptive neurons provides a comprehensive
anatomical basis for studies of the mechanisms through
which these neurons are controlled. For example, by monitoring the changes in noxious stimulus-evoked expression of
c-fos that are produced by systemic, intracerebral, or spinal
administration of narcotics, it should be possible to better
understand the relative contribution of spinal and supraspinal mechanisms to opiate analgesia (Presley et al., 88a).

ACKNOWLEDGMENTS
We thank Mme. Annie Menktrey and Ms. Simona Ikeda
for excellent help with graphics and photography. We also
thank Dr. Jan Tuttleman for providing some of the antisera
and for her helpful suggestions as to their use. This work was
supported by P H S grants NS14627, NS21445 and
AM32634. D. Menktrey is supported by the Centre National
de la Recherche Scientifique, France, and a fellowship from
NATO.

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